Aim To establish a rapid method to efficiently iso-late mononuclear cells from central nervous system ( CNS) tis-sues of mice that can be effectively utilized for identification of various immune cell populations in a single sample by flow cy-tometry. Methods For defining the feasibility and practicality of the method, wild-type C57BL/6 mice and two mouse models of CNS disease including EAE mice and APP/PS1 mice were used in this study. After the collection and homogenization of the brain and spinal cord tissues respectively, the mononuclear cells were isolated by spinning the 70% -30% Percoll gradients. Cell activities were detected by trypan blue staining, and the im-mune population that infiltrated CNS was identified by flow cy-tometry. Results The results of trypan blue staining showed that the survival rate of the isolated cells was above 90% in all groups. Flow cytometry analysis showed that the relative num- bers of lymphocytes infiltrating CNS of EAE and AD mice in-creased significantly compared with wild-type C57BL/6 mice. In addition, the relative numbers of Th1 and Th17 cell subsets me-diating the inflammatory response also increased significantly, while the decreased regulatory T cells frequency was observed in the two mouse models of CNS disease. Conclusions The cells isolated by the 70% ~30% Percoll gradients centrifugation can be effectively utilized for the identification of various immune cell populations in a single sample by flow cytometry. The meth-od described in this article is simple and rapid in operation and with high survival rate and activity of the cells, which can be ap-plied to the study of the mononuclear cells in CNS.

OBJECTIVETo preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis.

METHODSWistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria.

RESULTSThe sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria.

CONCLUSIONSIn the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

Animals ; Brain ; pathology ; physiopathology ; ultrastructure ; Lipopolysaccharides ; toxicity ; Male ; Membrane Potential, Mitochondrial ; Mitochondria ; physiology ; ultrastructure ; Rats ; Rats, Wistar ; Sepsis ; chemically induced ; mortality ; physiopathology

Objective:To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury.Methods:The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K + concentration was detected by FluxOR TMⅡgreen potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot. Results:Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1β, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1β (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K + concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K + concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K + massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages. Conclusion:Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K + efflux.

Objective:To observe the clinical efficacy of mouse nerve growth factor (mNGF) combined with rehabilitation on children with global developmental delay(GDD).Methods:It was a prospective multicenter clinical randomized controlled trial (RCT) involving 120 children with GDD admitted to 5 hospitals in China from May 2020 to January 2022.They were randomly divided into mNGF group and conventional rehabilitation group using block randomization method.All children were managed by standardized rehabilitation after recruitment, and those in the mNGF group were additionally given mNGF injections.All subjects were surveyed using the Gesell Development Diagnosis Schedules(GDDS) at baseline, 90 days and 120 days after treatment, and their developmental quotient (DQ) was recorded.Clinical efficacy was analyzed by the paired t-test, rank sum test and Chi- squared test. Results:After 90 days of treatment and the continuous follow-up to 120 days, the increases in the DQ of gross motor (7.520±13.900 vs.0.450±11.459), fine motor (7.800±15.346 vs.1.250±11.581), adaptive behavior (7.730±13.428 vs.2.100±12.022) and personal-social behavior (6.780±11.651 vs.1.780±10.120) than baseline were significantly higher in mNGF group than those of conventional rehabilitation group (all P<0.05). Serious adverse events and important drug-related medical events were not reported. Conclusions:mNGF combined with rehabilitation effectively enhances the development levels of gross motor, fine motor, adaptive behavior and personal-social behavior, and continuously improves the condition of GDD in children with a high safety.

Objective:To assess the efficacy and safety of cinepazide maleate injection in the treatment of patients with acute ischemic stroke.Methods:A multicenter, randomized, double-blind, placebo-controlled phase Ⅳ clinical trial, led by Peking Union Medical College Hospital, was conducted in 65 Hospitals in China. The efficacy of cinepazide maleate injection in patients with acute anterior circulation cerebral infarction with onset time of ≤48 hours, 7≤National Institute of Health stroke scale (NIHSS) score ≤25 was assessed from August 2016 to February 2019, using the proportion of modified Rankin scale (mRS) score≤1 and Barthel index (BI) score≤95 on day 14 as efficacy endpoint. The patients were divided into treatment group who were treated with cinepazide maleate injection and control group who were treated with placebo.Results:A total 937 patients were involved in the final efficacy analysis (466 in treatment group and 471 in control group). The proportion of subjects with mRS score≤1 on day 14 after treatment were higher in the treatment group than that in the control group (102/466(21.89%) vs76/471(16.14%)). Logistic regression analysis showed that patients treated with cinepazide maleate were significantly more likely to have a favorable outcome (mRS score≤1) than patients treated with placebo on day 14 ( OR=0.677, 95% CI 0.484-0.948 , P=0.023), and patients treated with cinepazide maleate were more likely to reach independence in activities of daily living (Barthel Index ≥95) than those treated with placebo on day 14 (125/466(26.82%) vs 91/471(19.32%); OR=0.632, 95% CI0.459-0.869, P=0.005). The rate of adverse events was similar between the treatment and control groups. Conclusion:The 14-day treatment with cinepazide maleate injection could reduce the degree of disability whereas did not increase the risk of adverse events.

Objective:To analyze the sexual relationship power and its influencing factors in married couples with single human immunodeficiency virus （HIV） positive spouse， and to provide theoretical support for the development of appropriate HIV prevention and control measures. Methods:Data of HIV single positive couples were collected from the database of AIDS epidemic. Social demographic characteristics， AIDS related knowledge， sexual behavior and sexual relationship power of HIV single positive couples were obtained by self-designed questionnaire. Current situation and influencing factors of sexual relationship power of the negative spouses HIV single positive couples' were analyzed. Results:Among 191 HIV negative spouses， 72.3% had not heard of AIDS， 3.1% had a history of HIV testing， and 38.2% had high sexual power. Single factor analysis found that men （χ² =12.227，P<0.001）， high income （χ² =4.227，P =0.040）， high education level （χ² =18.188，P<0.001）， frequent drinking （χ²= 6.104，P=0.013）， and high-level awareness of AIDS （χ²=10.827，P=0.013） had higher power of sexual relationship. Multivariate analysis showed that men and individuals with college education or above had higher sexual power， with OR （95% CI） 4.102 （1.757-9.578） and 7.403 （2.810-19.503）， respectively. Conclusion:The low awareness rate of AIDS knowledge and the disharmonious relationship between husband and wife will increase the risk of AIDS transmission in the spouses. Male， economic income， education level and AIDS awareness rate are the important influencing factors of sexual relationship power.